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...actin and myosin, and enzymes for transmitter synthesis. Retrograde transport is used for transport of...vivo, microinjection, pressure injection or iontophoresis (applying small electric currents via inserted...methods are naturally occurring viruses that infect, persist, and migrate within neurons, and that are also ... Not all measures of transport efficiency are consistent which may be caused by the diversity of techniques...are not fully understood and may be a form of transcytosis where the AAV particle is taken up on one side...cross the blood brain barrier by endothelial transcytosis (Foust et al., 2009; Merkel et al., 2017), and...Aschauer, D.F., Kreuz, S., and Rumpel, S. (2013). Analysis of transduction efficiency, tropism and axonal...

...systems that are widely present in bacteria. They consist of a toxin which can affect a variety of cellular...division”). The encoded toxin-antitoxin system consists of CcdB (toxin) and CcdA (antitoxin) where CcdB...sequences in RNA resulting in a blockage of protein synthesis. The antitoxin MazE, which binds and inhibits ... contains the mazF gene and a gene for Zeocin resistance (Figure 2). This MazF-ZeoR cassette is flanked...) contained within it by selecting for Zeocin resistance. Then, counter-selection with methanol medium...systems Because restriction modification systems consist of two enzymes that have opposing activities, it...the systems just described, the hok/sok systems consist of three components: Host killing (hok), a long-living...

... in every experiment-based investigation of a hypothesis: proper controls! For every experiment, an investigator...ability of the cells to express the protein.   In analysis of the result in Figure 1, an internal control...includes the aforementioned internal control and is consistent with the result in Figure 1 could look like this...associated with the independent variable should be consistent over time and from different preparations of ...replicate control is important, it speaks to the consistency of the reagents and hands performing the experiment...experiment, not the reproducibility or consistency of the observed phenotype associated with the independent...replicates control for the reproducibility and consistency of the observed phenotype associated with the...

.... Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene...lentiviral vector: 1. No risk of insertional mutagenesis Lentiviral vectors tend to integrate into transcriptionally...2002), thus increasing the risk for insertional mutagenesis. This integration could lead to an undesirable...harbor these unwanted interruptions. Insertional mutagenesis is especially concerning in clinical settings...acute lymphoblastic leukemia due to insertional mutagenesis (Howe et al., 2008). 2. Transient expression ...cells, as they avoid the risk of insertional mutagenesis while still allowing for stable expression of...Sander, J. D. (2013). High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells...

... (2016) Enhanced Rice Blast Resistance by CRISPR/Cas9-Targeted Mutagenesis of the ERF Transcription Factor...longer shelf life and the development of disease-resistant crops to increase agricultural yield (Wang et ...including commonly used model organisms like Arabidopsis and Medicago truncatula and several crop species...sequence and ~75 nucleotide scaffold sequence) is consistent between plants and other organisms, but the promoter...GOI) into plant cells. A) The system typically consists of a Ti plasmid, into which the GOI is inserted...2013) Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas...homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat Biotechnol 32:947–951 ...

...antibiotic resistance cassette and you aren’t introducing an additional antibiotic resistance cassette ...where the inserted DNA includes an antibiotic resistance marker. After transformation, plate the cells...into the plasmid backbone contains a kanamycin resistance cassette, you would plate the cells on kanamycin-containing...cells carrying plasmids that contain a kanamycin resistance cassette will grow. However, often times, the...often used for cloning in yeast since antibiotic resistance spontaneously arises in yeast. For example, Markus...occurring genes for cloning purposes (antibiotic resistance genes, toxin genes, and the sacB gene). By tapping...

...through a phase of crisis where they 'collect' a number of mutations.” Cystic fibrosis may not be the most...Cas9 genome editing system to correct the cystic fibrosis transmembrane conductor receptor (CFTR) by homologous... intestinal stem cells of patients with cystic fibrosis (8).   The difference with CRISPR/Cas9 in comparison...stem cells isolated from two patients with cystic fibrosis. They were able to demonstrate functional correction...said Gerald Schwank, first author of the cystic fibrosis report. “This is a big advantage over induced ...Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients." Cell Stem Cell. 13, 653-658 (5 December...

...salts. Capillary electrophoresis Quantifying DNA using capillary electrophoresis is similar to quantifying... using gel electrophoresis. Except, it’s smaller. And automated. Unlike gel electrophoresis, you only ... sample concentration for you. Agarose gel electrophoresis Though not the quickest way to quantify DNA...nanofluidic channel and the sample is separated by electrophoresis. Smaller fragments migrate quicker than larger...quantification of a RNA ladder using capillary electrophoresis which shows fluorescence units over time. Smaller...

...breakdown of the nuclear envelope, which occurs during mitosis. The most well-known retrovirus is HIV. Lentiviruses...the fear of random genomic integration causing mutagenesis in the host. Adenoviruses can deliver relatively...Dividing Cells Moderate Immunogenicity Insertional Mutagenesis Possible Lentivirus Infects non-dividing...non-dividing cells Insertional Mutagenesis Possible Adenovirus Remains Episomal (Little Risk of...of Insertional Mutagenesis) High Gene Expression Infects Non-Dividing Cells Large Packaging Capacity (... AAV Remains Episomal (Little Risk of Mutagenesis) Very Low Immunogenicity Infects Non-Dividing...Integration of murine leukemia virus DNA depends on mitosis.” EMBO J. 12 (1993) 2099-2108. PubMed PMID: 8491198...

...lab developed the Phage-Assisted Continuous Evolution (PACE) and Phage-Assisted Non-Continuous Evolution...Base editors make specific DNA base changes and consist of a catalytically impaired Cas protein (dCas or... to base editor activity and editing speed and consists of the following components: A plasmid in the.... A phage encoding the deaminase TadA-7.10 A mutagenesis plasmid controlling the phage mutation rate ...stringent phage selection. Figure 2: Phage-assisted evolution of base-editing activity. A plasmid-...Zeng J, Bauer DE, Doudna JA, Liu DR (2020) Phage-assisted evolution of an adenine base editor with improved...

...the heavy lifting, so don’t let the statistical analysis of your data get you hung up. Here we will dive... for you. Statistics – The basics Statistical analysis is what determines if there is statistical significance...account for subject-to-subject variation in its analysis (the test assesses the difference of the group...differences exist between. For example, if an ANOVA analysis on a five-sample data set yields statistical significance...pairings, additional tests, referred to as post-hoc analysis, must be performed. These will be discussed later...information. A post hoc test, done after your initial analysis can provide information on specifically which ...statistically different within your ANOVA/ANCOVA analysis. If you have performed a MANOVA/MANCOVA and want...

...insertional mutagenesis in humans (1) and AAVs are non-enveloped viruses, so they are resistant to alcohol-based... viruses, AAVs normally require second strand synthesis before gene expression begins. ScAAV avoid this... Russell, David W. "AAV vectors, insertional mutagenesis, and cancer." Molecular Therapy 15.10 (2007):...PMID: 17882145. 2. Zincarelli, Carmela, et al. "Analysis of AAV serotypes 1–9 mediated gene expression ...Dominik F., Sebastian Kreuz, and Simon Rumpel. "Analysis of transduction efficiency, tropism and axonal...promote efficient transduction independently of DNA synthesis." Gene therapy 8.16 (2001): 1248. PubMed PMID:...8. Ferrari, Forrest K., et al. "Second-strand synthesis is a rate-limiting step for efficient transduction...

...off-target analysis in order to meet clinical approval. It is key to maintain a consistent set of standards...connections can help alleviate the reproducibility crisis in biology. It is key to address these concerns... The results are then visualized using gel electrophoresis. Surveyor™ and T7E1 have been widely adopted...including Digenome-seq (Kim et al. 2015) for in vitro analysis, IDLV for in vivo detection (Gabriel et al. 2011..., Yanfang, et al. "High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells... sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic...Gabriel, Richard, et al. "An unbiased genome-wide analysis of zinc-finger nuclease specificity." Nature biotechnology...

...cytometry provides analysis at the cellular level, it cannot be used for subcellular analysis such as morphology...lymphocytes and “gate” that population for further analysis. Since different populations of immune cells express...around their desired cell population for further analysis. In this example, the lymphocyte population is...Include proper controls To ensure accurate data analysis, include a complete set of controls. Ideally, ...allow you to omit dead or dying cells from your analysis; dead or dying cells tend to have more autofluorescence... panel will help you set up gating during data analysis.  We hope that this blog has provided you with... an overview of flow cytometry for single cell analysis and some useful background information for setting...

...Here's what you'll find in this post: Peptide-assisted genome editing for primary cells Monomeric StayGold...Recombinant anti-FLAG tag antibodies Peptide-assisted genome editing for primary cells By Susanna Stroik...optimizing the Cas protein delivery with Peptide-Assisted Genome Editing (PAGE). They fused the Cas enzyme...cell-penetrating peptides and co-incubate it with an “assist peptide” that facilitates endosomal escape. Combining...mCherry reporter (mChe), with co-incubation with assist peptide (AP). C) Workflow for Cas-RNP-PAGE editing...primary T cells, by co-incubation of Cas-RNP and assist peptide. Adapted from Zhang et al. 2023 with permission...of human and mouse primary cells using peptide-assisted genome editing. Nature Biotechnology, 10.1038/...

...heating and cooling steps. PCR relies on heat resistant DNA polymerase from the thermophilic bacterium...aquaticus (Taq). Taq polymerase is thus a heat resistant enzyme that can withstand changes in temperature...generating mutated sequences for site-directed mutagenesis.  Quantitative polymerase chain reaction, qPCR... high throughput SNP genotyping, gene deletion analysis, and RNA detection.  A summary of the types of...interest  Molecular Cloning Site-directed mutagenesis Molecular Diagnostics qPCR Quantify starting...pathogen identification SNP genotyping Gene deletion Analysis RNA detection     References and Resources... PCR  Plasmids 101: Colony PCR Site Directed Mutagenesis by PCR Resources on Addgene.org Protocol: Polymerase...

...newly-deaminated inosines. Pro tip! The nucleoside inosine consists of the nucleobase hypoxanthine + a ribose sugar...this promising start, the team used rational mutagenesis to produce the adenine transversion editor AYBEv3...editing, they co-expressed Polη, a translesion synthesis polymerase known to preferentially incorporate...and ABEs, they could be useful for saturation mutagenesis, and they provide a strong proof of concept for... editor (gGBE) (Tong, Liu, et al., 2023). gGBE consists only of engineered MPG fused to nCas9. First, ...empty space. Over several rounds of rational mutagenesis, the team progressed to gGBEv6.3, which was capable...., & Chang, X. (2024). Protein language models-assisted optimization of a uracil-N-glycosylase variant...

...expression levels comparable to GFP during early embryogenesis (Figure 3). mScarlet-I3 also offered clearer... Pe K., Cesar M., Hobert O. (2024) Comparative analysis of new mScarlet-based red fluorescent tags in ... common human pathogen notorious for its high resistance to antibiotics. Several CRISPR/Cas-based systems...abscessus’ high rate of spontaneous antibiotic resistance interfering with selection. Using an inducible...understanding of its biology, pathogenicity, and resistance mechanisms.     Figure 4. Overview of...plasmid containing tetracycline repressor TetR and resistance selection marker KanR. This strain is then transformed... cassette on a plasmid expressing mCherry and resistance selection marker HygR. The CRISPR system is induced...

...codon usage important? Translation and protein synthesis relies on transfer RNAs (tRNAs). tRNAs bind to...2010). Figure 1: An overview of peptide synthesis. A ribosome interacts with mRNA and charged tRNAs... altering the sequence of the protein. As DNA synthesis has become quite cost-effective it seems easy ...algorithms/resources (Athey et al., 2017) and many DNA synthesis companies, such as IDT, Genewiz and GenScript,...system of choice. ThermoFischers GeneArt Gene Synthesis NovoPro Labs Codon Optimization Tool (ExoOptimizer... Hausser J, Syed AP, Bilen B, Zavolan M (2013) Analysis of CDS-located miRNA target sites suggests that....139758.112 Mauro VP, Chappell SA (2014) A critical analysis of codon optimization in human therapeutics. Trends...

...phosphatase inhibitors to your lysis buffer. Additionally, protein lysis should be done at 4 °C or on ice...conditions Adjust lysis conditions: time, temperature, and/or buffer to increase lysis Add sonication...temperature. Some proteins aggregate at 95 °C, a common lysis temperature, so consider a longer incubation (10...ladder on the lefthand side. Whatever you do, be consistent and document it.   Did it work? Save yourself...chemical buffer around, which works well and consistently, to help troubleshoot background issues. It’...sonication step to protein lysis Splice variants Check literature Posttranslational modifications ...Run control protein (if available) and note in analysis Plasmid-based proteins: plasmid sequence not...

...cell cycle progression, or apoptosis with Cas9/gRNA expression. A pooled analysis found no evidence of Cas9... HIV-1 toxic proteins, CRISPR/Cas9 SAM caused apoptosis, indicating a true “shock and kill” response. ...binding. It’s also possible that HIV-1 may evolve resistance to CRISPR therapies through PAM or seed sequence...multiple drugs to lower the odds of developing resistance. Shortly after Kaminski et al. published their...not ablate viral function, but rather promote resistance to CRISPR/Cas9. While these results are discouraging...synergistic activation mediator (SAM) induces specific, persistent and robust reactivation of the HIV-1 latent reservoirs...

...generate additional recombination events, and the synthesis of alternative loxP sites to specify distinct ...schematic at the bottom of this post for a basic synopsis of the differences amongst the Brainbow systems...neurons’ somata. Second, the system did not permit analysis by immunostaining. Although the fluorophores used...cross-reactivity, opening the door for immunostaining analysis. To achieve even cell labeling, they generated.... Epub 2008 Apr 30. PubMed. Intestinal crypt homeostasis results from neutral competition between symmetrically...genetic multicolor cell labeling for neural circuit analysis in Drosophila melanogaster. Hadjieconomou D, Rotkopf...multispectral cell labeling for cell tracing and lineage analysis in zebrafish. Pan YA, Freundlich T, Weissman TA...

...genes chosen from an analysis of human metastasis cohorts and a prior mouse metastasis CRISPR screen. The...discovered a new Cas protein, CasX, from metagenomic analysis of microbial DNA from groundwater. Now, they’ve...knockout libraries for cancer research. The Mouse Metastasis CRISPR-Cpf1(Cas12a) Double Knockout Library is...tumor suppressor genes (TSG). Find the Mouse Metastasis CRISPR-Cpf1 Double Knockout Library Find the...

...continue to improve plant nutrition, yield, and pest resistance. Much of plant genetic engineering revolves around...functions in multiple species, including tobacco and Arabidopsis. Improving Agrobacterium binary vectors Vectors...domains and characterized in studies of tobacco, Arabidopsis, and corn. For both β-glucuronidase (GUS) and...efficiently inherited in both transgenic tobacco and Arabidopsis. Finally, Sahoo et al. demonstrate that this ...variety of plant species. The kanamycin antibiotic resistance gene is controlled by a weaker constitutive Nos...Plant Synthetic Biology Quick, Versatile Plant Transgenesis with GreenGate Plasmids Interview: Nicola Patron...

...should be consistent across all samples on the gel and should be observed as a band of consistent size and... Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is the first step of a western. ...separate by size. Smaller proteins encounter less resistance in the acrylamide matrix and migrate faster through...percentage of acrylamide to increase migration resistance and improve separation. For large proteins, reduce...it’s time to analyze your protein of interest. Analysis typically begins by confirming that the protein...loading or incomplete protein transfer causes inconsistent levels of the loading control. In these cases...

...data analysis/visualization using R/Shiny. She is passionate about open science, BIG data analysis and ...interpretations. A good peer-reviewer will help spot inconsistencies in the work, missing information that would... specific repositories (Addgene, WormBase, Arabidopsis.info). These repositories will ensure that the ...induced responses in Root System Architecture of Arabidopsis HapMap population in Amsterdam. Currently, she...salt-induced changes in root-to-shoot ratio in Arabidopsis, (2) expression patterns in plants with enhanced...

...fact, due to impaired endocytosis. In parallel assays designed to test the endocytosis of a fluorescent dye...strategies or developing novel methods for data analysis, our team’s goal is to provide scientists with...cell membrane and are taken into the cell via endocytosis. In our study, we aimed to prevent the uptake...packaging cells by treating the cells with the endocytosis inhibitor sucrose. Previous work by Stoneham ...Hollinshead, and Amin Hajitou. "Clathrin-mediated endocytosis and subsequent endo-lysosomal trafficking of ...

...Silver.  Most types of biological motion (whether endocytosis, vesicle trafficking, or muscle contractions)...video. Most types of biological motion (whether endocytosis, vesicle trafficking, or muscle contractions)...studied for many decades, and they were known to be resistant to detergents and degradation under a variety ...demonstrated that R bodies can be “tuned” with mutagenesis to extend at different pH values. Using a screen...in a particular region of the R-body. Further mutatgenesis of this region produced R-bodies that could ...Organization and expression of genetic determinants for synthesis and assembly of type 51 R bodies. J. Bacteriol...

...for experience in “statistical analysis”, mention “statistical analysis” (word by word) in your resume...large data sets and are familiar with statistical analysis. If you have done fluorescent imaging, then you...you probably have experience using image analysis software. If you use computer programs to predict protein...universities even have officers working specifically to assist graduate students in job searching. Start your ...resume writing, as well as additional videos with emphasis for graduate student resumes UC Irvine Non Academic...

...mitochondrial homeostasis maintained through mechanisms like fission, fusion, biogenesis, and mitophagy...proteins are SNAP-tag® and HaloTag®. These tags consist of a genetically encoded enzyme variant that covalently...photostability is crucial. These properties ensure a more consistent and stable signal over extended periods, which...clear differentiation between multiple dyes during analysis. Image resolution is impacted by several factors...fluorescence. When it comes to protein labeling and analysis, dyes should be small enough not to hinder protein...focuses on commercializing their cell and protein analysis products. You can find plasmids deposited by Promega...

...biology in generalized pustular psoriasis Generalized pustular psoriasis is a chronic autoinflammatory ...says Iyer. Since the RESOLUTE plasmid collection consists of human and murine solute channels, the team ...in the biology of GPCRs and G proteins, with an emphasis on oligomerization, signalling complex assembly...nucleus,” says Dr. Terry Hébert, a professor and assistant dean of biomedical science education at McGill... University.  These protein-receptor complexes consist of many parts that work together on the cell membrane...

...workflow and its analysis can be found here: A Novel Next-Generation Sequencing and Analysis Platform to Assess... example, the CAG promoter, a hybrid promoter consisting of a CMV enhancer, chicken beta actin promoter...the FASTQ data from our instrument and begin the analysis. We use a modified version of the Python program...Ersing I, Haery L, Harten DeMaio K, Sanders E, Tasissa M, Kostman M, Tillgren M, Makana Hanley L, Mueller... (2020) A Novel Next-Generation Sequencing and Analysis Platform to Assess the Identity of Recombinant...

...the infants. Almost all patients have achieved persistent immunological reconstitution with a normally ...disease-associated mutations. AAV vectors maintain persistent transgene expression over many years in postmitotic...limited to hemophilia B, LPL deficiency, Cystic fibrosis, Muscular Dystrophy, Parkinson’s disease, and ... form of blindness known as Leber’s congenital amurosis (LCA). This disease is caused by mutations in ... the human gene RPE65, patients with clinical diagnosis of LCA and mutations in RPE65 (n=3), showed significant...Furthermore, deeper understanding of disease pathogenesis will enable robust therapeutic outcomes. In ...

...carbohydrates that can inhibit the cell lysis process. To ensure that lysis is complete, use a reagent like Qiagen...your plasmid yield is sub-optimal? If you are consistently getting sub-optimal plasmid yields from your...Addgene uses and recommends DH5𝝰! This strain consistently gives good, high quality preps due to its endA1...Qiagen’s LyseBlue, which uses color to show you if lysis is incomplete. Protein expression strains are not...produce fewer transcripts of their antibiotic resistance genes and can therefore be cultured in media ...

...is its simplicity and versatility. CRISPR/Cas9 consists of two components: the non-specific endonuclease...libraries (referred to simply as “CRISPR libraries”) consist of a heterogeneous population of gRNA-containing...drug screens to identify genes that confer drug-resistance. Mutant cells are treated with a drug of interest...and gRNA distribution is analyzed in the drug-resistant population compared to a non-treated control group...enriched” correspond to genes that confer drug resistance when mutated. Findings from this type of experiment...shed light on the mechanism by which cells gain resistance to drugs and can identify future therapeutic ...

... to the 5’ end of the reverse primer. Mutagenesis - Mutagenesis is performed by designing primers astride...modification, from insertions, deletions, and point mutagenesis to complex assembly of multiple genes. Primer...are due to incorrect primer sequences! Primers consist of two regions: the 3’ end binds template DNA for...unamplifiable’ Check your amplification by gel electrophoresis. In our experience, good amplification after...rapid method for recombination and site-specific mutagenesis by placing homologous ends on DNA using polymerase...

...Agarose Gel Electrophoresis Inoculating Liquid Bacterial Cultures Restriction Digest Analysis We also...fastest shipping. However, there were issues that persisted despite our best efforts. With a little over half...having some materials available to order, yet not consistently accessible, wasn’t an option. So we identified...γ-Retrovirus Guide Protocols Agarose Gel Electrophoresis Bacterial Transformation How to Design a Primer...Verify Your Plasmid Using a Restriction Digest Analysis Videos What is a Plasmid? How to Design...

...incompatibility with antibiotic-resistance plasmids, scientists have expelled antibiotic resistant plasmids from Enterobacteriaceae...partitioning system in the bacteria, but generally it consists of a centromere-like region on the plasmid, a ...plasmid and pair the plasmids together (akin to sister chromatids coming together in eukaryotic cells)...plasmid incompatibility to combat antimicrobial resistance. Using novel plasmids based partly on plasmid...2017) Plasmid interference for curing antibiotic resistance plasmids in vivo. PLOS ONE 12:e0172913 . https...

...method for transient gene expression analysis in seedlings of Arabidopsis and other plant species." Plant ...   C. roseus is not a model organism such as Arabidopsis or tobacco. Studying gene function in C. roseus...method for overexpressing genes In plants like Arabidopsis and tobacco, Agrobacterium infiltration into ...expression assay by agroinfiltration of leaves." Arabidopsis Protocols. Humana Press, 2006. 225-229. PubMed...of our plant biology blog posts Find tips for Arabidopsis transformation Read our Plasmids 101 blog posts...

...Efficient and Stable Genome integration Insertional mutagenesis, transgene reactivation, residual expression ...Efficient and Stable Genome integration Insertional mutagenesis, residual expression piggyBac Excisible Medium...generating methods due to the risk of insertional mutagenesis. Random integration also creates heterogeneous...Finally, retroviruses are prone to insertional mutagenesis and random integration creates variation between...because they are less likely to cause insertional mutagenesis, adenoviral vectors are considered a safer way...These methods eliminate the risk of insertional mutagenesis, the presence of a genetic scar, and incomplete...replication of the plasmid in mammalian cells and assists in tethering the vector to the cell’s chromosomes...